Abstract Considerable evidence has implicated the protein -synuclein in the pathogenesis of Parkinson's disease (PD). Point mutations in -synuclein can cause autosomal dominant PD, and -synuclein accumulates in the Lewy bodies and dystrophic neurites of sporadic PD, suggesting a role for the protein in most forms of the disorder. Importantly, an increased dose of the wild type gene can also cause PD, indicating a pathogenic role for the normal protein and perhaps its normal function. However, the role of -synuclein in PD and its normal function remain poorly understood. Studies in yeast and mammalian systems have suggested a role for -synuclein in membrane trafficking. The protein interacts with membranes in vitro and localizes primarily to the axon terminal in neurons, but its effects on synaptic vesicle exocytosis and recycling remain controversial and poorly understood. The long-term objective of this proposal is to elucidate the function of -synuclein in its physiologically relevant context, at the nerve terminal. Since PD seems to involve an increase in -synuclein, the strategy has been to over-express the protein in primary neuronal culture, and assess its effects on the synaptic vesicle cycle by optical imaging of live cells. In preliminary experiments using a combination of optical imaging, electrophysiology, biochemistry and electron microscopy, we have found that over-expression of -synuclein impairs neurotransmitter release by reducing the size of the synaptic vesicle recycling pool, providing some of the first unambiguous evidence for its role in neurons. We now propose to: 1) determine whether the inhibition of transmitter release by synuclein involves a gain in its normal function, by studying triple synuclein knock-out mice; 2) characterize the effect of - synuclein on dispersion and reclustering of synaptic vesicle protein and membrane after exocytosis; 3) assess the relationship between synuclein, synapsins and synphilin in synaptic vesicle mobilization; and 4) determine whether the effects of synuclein on neurotransmitter release contribute to degeneration. The results will extend previous work on -synuclein to its appropriate biological context in neurons, and provide a crucial physiological framework to understand the function of - synuclein. In the process, they will address a mechanism that may give rise to the degeneration observed in Parkinson's disease, and suggest therapeutic strategies to reverse functional disability due to the impaired release of transmitter from neurons that survive.